Mutant <scp>NPM1</scp>: Nuclear export and the mechanism of leukemogenesis
Authors: Brunangelo Falini, Maria Paola Martelli, Lorenzo Brunetti
Journal: American Journal of Hematology (2023)

NPM1-mutated acute myeloid leukemia (AML) represents the most frequent genetic subtype of AML, accounting for about 30% to 35% of newly diagnosed cases in adults.1 Because of its distinctive clinico-pathological and molecular features,2-4 NPM1-mutated AML is recognized as a distinct entity in the World Health Organization (WHO) classification of myeloid neoplasms.5, 6 The NPM1 gene encodes for a multifunctional protein with chaperone activity,7 which continuously shuttles between the nucleus and the cytoplasm, but mainly resides in the nucleoli.8 The most distinguishing feature of NPM1-mutated AML is the aberrant localization of mutant NPM1 (NPM1c) from the nucleolus to the cytoplasm of leukemic cells.9-11 The abnormal NPM1c localization is secondary to the loss of nucleolar localization signal (tryptophans at positions 288 and 290 or 290 only) and the acquisition of a strong nuclear export signal at the C-terminus, which promotes the interaction with the nuclear exporter Exportin-1 (XPO1) and results in the cytoplasmic accumulation of mutant NPM1.10 Another unique feature of NPM1-mutated AML is the high expression of homeobox (HOX) transcription factors.2 More specifically, HOXA and HOXB genes, as well as their cofactor MEIS1 (hereafter referred as to HOX/MEIS), are always detected at high levels in NPM1-mutated cells.2 Although HOX/MEIS have been regarded as critical players for AML development, their essential role in the maintenance of NPM1-mutated AML and their direct dependency on NPM1c have been demonstrated only recently.12 Only limited information is currently available on the precise mechanism(s) through which NPM1c leads to AML development. The pronounced nuclear export and cytoplasmic accumulation of NPM1c have been hypothesized to be involved in AML pathogenesis.4 On the other hand, the link between the cytoplasmic accumulation of the NPM1c oncoprotein and the transcription of HOX/MEIS, which takes place in the nucleus, has been a matter of debate.4 The concept that NPM1c may exert its activity in the nucleus is supported by nucleocytoplasmic fractionation experiments in the NPM1-mutated OCI-AML3 cell line showing that about 10% of the mutant protein is retained in the nucleus while the remainder is delocalized into the cytoplasm.13 This is also confirmed by immunohistochemistry studies on bone marrow biopsies from NPM1-mutated AML patients performed with a specific antibody that recognizes NPM1c but not wild-type NPM1, revealing that, in addition to the cytoplasm, NPM1c can be detected at low levels in the nucleus (Falini B, unpublished results). Such nuclear localization is not surprising since NPM1c is known to contain a bipartite nuclear localization signal (NLS) that can drive it from the cytoplasm back to the nucleus.10 Two compound papers published in Cancer Discovery in December 2022 have now established that NPM1c drives HOX/MEIS expression directly from inside the nucleus. Using chromatin immunoprecipitation13 and CUT&RUN techniques,14 both studies demonstrate that NPM1c binds to chromatin. While Uckelmann et al.13 show that NPM1c is particularly enriched at a limited number of genes, all of which are highly dependent on NPM1c for their transcription, Wang et al.14 detected NPM1c at a much larger number of targets. Regardless of this dissimilarity, which could be secondary to differences in the experimental procedures and bioinformatic analysis, both studies clearly show that NPM1c is recruited at HOX/MEIS and directly regulates the transcription of these critical targets. Indeed, NPM1c was found enriched at active chromatin sites, together with activating histone marks, MLL1 and RNA Polymerase II (Pol II) and specific degradation of NPM1c resulted in immediate halt of HOX/MEIS transcription (within 15 min) secondary to the loss of Pol II from these loci.13, 14 Furthermore, pharmacologic MLL-Menin inhibition induced the loss of NPM1c from several of its chromatin targets, including MEIS1, followed by their downregulation.13 How is NPM1c recruited to chromatin? Both studies suggest that XPO1 is critical for NPM1c enrichment at HOX/MEIS. Specifically, pharmacologic inhibition of the interaction between NPM1c and XPO1 invariably results in the release of NPM1c from its targets, followed by loss of HOX/MEIS expression. In this regard, XPO1 has already been proposed to recruit other oncoproteins to chromatin, particularly NUP98 fusion proteins,15 which are known to drive HOX/MEIS expression in AML. This suggests that NUP98 rearranged leukemias and NPM1-mutated AML may share some features in their molecular pathogenesis. In addition to XPO1, Uckelmann et al.13 demonstrated that one of the two NPM1 acidic domains (~aa 120 to 150) is also necessary for an efficient NPM1c recruitment to chromatin. Wang et al.14 proposed that the generation of NPM1c phase separated nuclear condensates is critical for the recruitment of NPM1c to chromatin and to hijack the transcriptional machinery at its target loci. Whether NPM1c condensates are actually necessary for HOX/MEIS expression and whether the acidic domain is involved in the process of phase separation process itself remains to be clarified. NPM1c oncogenic activity at the chromatin level may seem counterintuitive if we accept that NPM1c nuclear export and cytoplasmic accumulation are relevant processes for AML development and maintenance. However, if NPM1c promoted leukemogenesis by simultaneously acting in the nucleus and in the cytoplasm, it would operate by "killing two birds with one stone" (see Figure 1). The strongest indirect evidence of the importance of nuclear export in the pathogenesis of NPM1-mutated AML is that all NPM1 mutants described so far, including those rarely affecting exons other than 12, for example, exons 516 and 11,17 invariably lead to cytoplasmic accumulation of NPM1 mutants. In addition, the nuclear export of NPM1 mutants is fine-tuned by the strength of the C-terminal NES, that is insertion of a weaker NES (when both tryptophans 280 and 290 are lost) or insertion of a stronger NES (when only tryptophan 290 is lost).10, 18 Thus, all NPM1 mutations are "born to be exported", that is, they act maximizing the interaction with XPO1 and the export and cytoplasmic accumulation of NPM1 mutants.18 Since the nuclear export is an energy-consuming process, cytoplasmic accumulation of NPM1c would be detrimental if not finalized to increase cellular fitness and promote leukemic transformation. Based on available data, the cytoplasmic fraction of NPM1c could promote AML development through at least two neomorphic mechanisms: (1) binding and relocating nuclear proteins to the cytoplasm, and (2) binding directly to cytoplasmic proteins. In both cases, NPM1c would interfere with their physiological function, promoting AML development. As for the first option, NPM1c has been implicated in the cytoplasmic delocalization of several nuclear proteins involved in apoptosis, DNA repair, and differentiation, including ARF, HEXIM1, FBW7, MIZ1, HEXIM1, CTCF and PU.1.10, 19-23 Although the exact proportion and impact of the cytoplasmic relocation of these proteins remain to be established,10, 24 it is predicted that interfering with biological processes critical for hematopoietic progenitors such as apoptosis and differentiation would support AML development. In contrast with the previous mechanism, direct evidence that NPM1c can directly interact with proteins physiologically located within the cytoplasm is limited to only one report. Specifically, NPM1c can inhibit the activities of the cell-death proteases, caspase-6 and -8, through direct interaction with their active forms, resulting into protection from death ligand-induced cell death and suppression of caspase-6/−8–mediated myeloid differentiation.25 Such limited information available on possible neomorphic functions of NPM1c in the cytoplasm should encourage us to keep searching in this direction, since at present we are likely looking only at the tip of the iceberg. In conclusion, we hypothesize that NPM1c promotes AML development and maintenance by acting both in the nucleus and in the cytoplasm. This hypothesis not only has potential biological implications but also implies that drugs interfering with the NPM1c-chromatin interaction (e.g., Menin and XPO1 inhibitors)26 and blocking the nuclear export of NPM1c (e.g., XPO1 inhibitors)27 should be further tested in NPM1-mutated AML patients, both as single agents and in combinations. Brunangelo Falini had the original idea. All authors contributed to write the article. Open Access Funding provided by Universita degli Studi di Perugia within the CRUI-CARE Agreement. BF licensed a patent on NPM1 mutants (n. 102 004 901 256 449) and declares honoraria from Rasna Therapeutics, Inc for scientific advisor activities. BF is a member in the Neogenomics scientific advisory board. MPM declares honoraria from Rasna Therapeutics, Inc for scientific advisor activities and honoraria/consultancy at scientific advisory board for Abbvie, Amgen, Celgene, Janssen, Novartis, Pfizer, Jazz Pharmaceuticals. LB has no relevant conflict of interest. Data sharing is not applicable to this article as no new data were created or analyzed in this study.